The death receptor Fas (CD95/APO-1) mediates apoptosis in many tissues. Upon stimulation by its ligand FasL, it forms a death inducing signaling complex (DISC) consisting of the adaptor molecule FADD, the initiator caspases 8 and 10, and the caspase-8/10 regulator c-FLIP. Recently it has become clear that, in addition to its role as a death receptor, Fas has multiple nonapoptotic activities including a role in rendering Fas apoptosis-resistant cancer cells more mobile and invasive. These novel activities also rely on DISC components, raising the question of how the choice to engage apoptotic or nonapoptotic signaling pathways is made. We have recently shown that Fas requires internalization into an endosomal/lysosomal compartment in order to signal apoptosis. However, in striking contrast, we found that receptor internalization is not required for nonapoptotic signaling through Fas. We found that tyrosine 291 is involved in regulating the internalization of Fas and that another postranslational modification, palmitoylation of cysteine 199, facilitates the formation of high molecular weight DISC complexes (hiDISC). hiDISC forms at the sites of Fas internalization. Recently, we found that Fas suppresses the expression of the microRNA let-7 and we obtained evidence that let-7 drives tumor progression through targeting HMGA2 and IMP-1. We hypothesize that the two posttranslational modifications of Fas determine whether Fas is internalized and whether it activates apoptotic or nonapoptotic signaling pathways resulting in downregulation of let-7. Identification of the role and the mechanisms of the internalization of Fas, its regulation by posttranslational modifications, and the regulation of let-7 expression by Fas could provide the means to neutralize the tumor promoting activities of Fas. We propose the following three specific aims: Aim 1: Determine the mechanisms that regulate whether Fas signals apoptosis or survival. Aim 2: Characterize the functional connection between Fas and let-7. Aim 3: Elucidate the function of Fas signaling mutants and of Fas regulated miRNAs in mouse models of ovarian cancer. Characterization of the novel mechanisms and functional consequences of nonapoptotic signaling of Fas may facilitate the development of rational strategies and therapeutic approaches for interfering with dysregulated Fas signaling in cancer and other diseases. PUBLIC HEALTH RELEVANCE: The death receptor Fas (CD95/APO-1) has been viewed as a mediator of apoptosis in many tissues but it has become clear that Fas has multiple nonapoptotic activities including the induction of proliferation and tumor invasiveness. We will study the early events of Fas signaling including receptor internalization that determine whether Fas signals apoptotic or nonapoptotic pathways and downregulation of tumor suppressing microRNAs. These studies will provide a basis for understanding the different signaling activities of Fas and to devise new therapeutic approaches for interfering with dysregulated Fas signaling in cancer and other diseases.